PROJECT SUMMARY Candida albicans is an oral commensal organism and an opportunistic pathogen. C. albicans is a frequent cause of oral infection in patients with compromised immune systems, including AIDS patients and patients undergoing chemotherapy, and in patients who produce low levels of saliva. To help prevent the burden of C. albicans from becoming too high, healthy individuals produce several immune molecules in their saliva. One of the most important of these molecules is histatin 5 (Hst-5), a member of the histatin family of histidine-rich antimicrobial peptides. Hst-5 is a 24-amino acid peptide and has the strongest candidacidal activity of the histatin peptides. Although salivary histatin is effective at killing C. albicans cells in the oral cavity, the pathogen also has a mechanism for evading killing by Hst-5 and other peptides. C. albicans produces a family of secreted and cell-wall anchored proteases called secreted aspartic proteases (Saps) that are capable of degrading Hst-5 and making it inactive. Previous work showed that Sap enzymes cleave Hst-5 at lysine residues, which is also observed in cleavage by C. albicans cells. Additionally, human saliva contains human and microbial proteases that can also degrade Hst-5. To reduce proteolytic cleavage of Hst-5 and improving its potential as a therapeutic in the oral environment, we designed several Hst-5 variants with the lysine residues substituted with a leucine or arginine residue. Initial proteolysis testing showed variants with resistance to cleavage by Sap2 and Sap9 enzymes and variants with amplified cleavage, highlighting the complexity of the interaction of the Saps with Hst-5. The modifications in the variants shifted the location of cleavage by the Sap enzymes and altered the antifungal activity of the degraded peptides. Importantly, peptides with improved resistance to cleavage maintained significant antifungal activity following incubation with the Sap enzymes. The substitutions generally did not result in loss of antifungal activity for the intact peptide, and several peptides exhibited stronger activity. To better understand the complex interaction between the Hst-5 and proteolytic enzymes in the oral cavity and enable design of improved peptide therapeutics, we propose to complete two aims in this work. The first aim is to characterize the interaction of salivary proteases and additional Saps with Hst-5 variants to understand the structure-function relationships that lead to cleavage (and lack of cleavage) and antifungal activity. The second aim is to use the knowledge gained through our preliminary data and Aim 1 to design and test a second generation of Hst-5 variants that further explore the effect of peptide sequence on proteolytic susceptibility. This project will improve our understanding of the interaction of Hst-5 with C. albicans Sap enzymes and enzymes in human saliva, providing the necessary knowledge to design peptides that could function as therapeutics to treat or prevent oral candidiasis.